Pack 15 - Recombinant DNA technology

Producing DNA fragments
1. A gene is a section of DNA on a chromosome coding for one or more polypeptides and some forms of RNA
2. Recombinant is the combining of DNA together from two different organisms
3. Major ways fragments of DNA can be produced:
  1. 1) conversion of mRNA to cDNA
  2. 2) Restriction endonuclease enzymes cutting from mRNA
  3. 3) Created in the "gene machine"
4. Process of GMO:
  1. 1) Isolation of the gene of interest
  2. 2) Insertion of gene
  3. 3) Transformation, transfer gene and vector into host
  4. 4) Identification of the host cells that have taken up the gene
  5. 5) Growth/cloning of the host cells
5. It is difficult to transfer genes from eukaryotes into prokaryotes due to introns (non-coding DNA)
  1. This can be overcome by using reverse transcriptase:
    1. This is the formation of DNA using RNA template
      1. Produces cDNA (no introns) which is:
        Complimentary DNA
        Made of DNA complementary to original mRNA
    2. Process of making cDNA:
      1. mRNA coding for a particular gene
        mRNA acts as template which single stranded DNA, cDNA formed with reverse transcriptase
        DNA is isolated by hydrolysis of RNA with an enzyme
        Double stranded DNA is formed by incubating with DNA polymerase and DNA nucleotides
6. Using restriction endonucleases
  1. These are a group of enzymes which cut the DNA at certain specific base sequences.
  2. Recognition sequences are often 6 bp long and are palindromic
  3. When cut, DNA can form one of two things:
    1. sticky end - single stranded over hang
    2. Blunt end - no single stranded overhang
      1. Sticky ends are better than blunt ends because blunt end is non-specific and can go in the wrong way round
7. Making a gene in a gene machine:
  1. 1) desired nucleotide sequence inputed into computer
    1. 2) computer designs series of small overlapping DNA sequences
      1. 3) Small single stranded sequences assembled by adding a nucleotide at a time
        3) join together to make gene
        4) Gene replication by PCR
        5) Genes are sequenced and those with errors are refected
        6) Correct genes are transferred to host cell
8. Preparing a DNA fragment for insertion into host cell
  1. Promoter and terminator required
    1. Promoter =
      1. Located in front of gene
      2. Specific sequence for binding of transcription factors
    2. Terminator =
      1. Location where DNA polymerase disengages from DNA
  2. Place a promoter in front of the gene and a terminator behind the gene
In vivo cloning - Use of vectors
1. In vivo = performed or taking placed in a living organism
2. In vitro = in glass
3. A vector is something which carries DNA into a cell
4. How to add DNA fragment into a Vector:
  1. 1) cut vector with sticky ends using restriction endonucleases
    1. 2) Cut the insert with sticky ends with restriction endonucleases
      1. 3) mix together
        4) single stranded DNA overhands complementary base pairs
        5) add DNA ligase to connect sugar-phosphate backbone on both strands
5. Advantages:
  1. Once DNA is in vector, it is easy to transform
  2. only gene complementary to sticky ends cloned
  3. Clone specific genes
  4. bacteria make multiple copes of gene
Introduction of DNA into host cells
1. By inserting the DNA into the host cell, every time it replicates it will have the DNA
2. Process of insertion is called transformation
3. Transformation can only occur when:
  1. Host bacterial cell is mixed with calcium ions to make them more permeable
  2. Chill in ice, then warmed to 42 degrees C
4. After transformation, not all bacteria take up the DNA because:
  1. Some plasmids may not contain the insert at all
  2. Sometimes DNA fragments join together to form plasmids
Screening for genetically modified cells
1. 1) Antibiotic resistance Marker Genes
  1. Cut plasmid with same RE used to produce gene fragment
    1. Add fragment and DNA ligase to cut plasmid
      1. Everywhere the ampicillin resistance gene goes, the gene of interest goes too, as they are joined together
        Recombinant plasmid
  2. When diluted and plated onto agar plates containing ampicillin, the two plates marked will die
    1. This leaves only the plate with the recombinant plasmid in, so these can be grown and used
2. 2) Fluorescence Marker Genes
  1. Screening for loss of fluorescence:
    1. 1) Original plasmid contains both antibiotic resistance and fluorescence protein gene
      1. 2) Gene of interest is inserted into centre of fluorescence gene
        3) diluted bacteria spread onto agar plate containing Ampicillin
        4) bacteria that do not glow green are selected
  2. Screening for gain of fluorescence:
    1. 1) Original plasmid contains only antibiotic resistance gene (Ampicillin)
      1. 2) the gene of interest together with the fluorescence gene is ligated into a plasmid
        3) Diluted bacteria are spread onto agar plate containing Ampicillin
        4) Bacteria that glow green are selected
3. 3) Enzyme Marker Genes
  1. Screening for loss of enzyme:
    1. 1) original plasmid contains both antibiotic resistance (Ampicillin) and LacZ enzyme gene
      1. 2) The gene of interest is inserted into the centre of LacZ gene
        3) Diluted bacteria are spread onto an agar plate containing Ampcillin and substrate
        4) Bacteria that do not hydrolyse the substrate are selected
  2. Screening for gain of enzyme:
    1. 1) original plasmid contains only an antibiotic resistance gene for Ampicillin
      1. 2) gene of interest is inserted together with LacZ
        3) Diluted bacteria are spread onto agar plate containing Ampicillin and substrate
        4) Bacteria that hydrolyse the substrate are selected
RE = restriction endonuclease
In Vitro gene cloning PCR
1. To replicate DNA in vitro you need:
  1. DNA fragment
  2. pair of primers
  3. Nucleotides
  4. DNA polymerase
  5. Thermocycler
2. How PCR works:
  1. 1) Strand separation - DNA heated to 95 degrees to break H bonds
    1. 2) Primer binding - cooled 55 degrees to allow primer to anneal
      1. 3) Strand synthesis - heat it to 72 degrees DNA polymerase, two strands of DNA complementary to original DNA
3. Advantages:
  1. make a lot of DNA
  2. Fast
  3. can be used for forensic analysis
  4. No need for culture cells
4. Disadvantages:
  1. Risk of contamination
  2. Risk of mutation
Relating recombinant DNA technology to gene therapy
1. Somatic cells = non-repoductive
  1. Germ cells = reproductive cells
2. Somatic cell therapy targets tissue
  1. Germ cell therapy occurs in fertilised eggs
3. Cystic Fibrosis
  1. Caused by deletion mutation
  2. Chlorine ions are kept inside the cell so osmosis cannot occur as well, less water will leave the cell
    1. Therefore, mucus builds up so infections cannot be removed
  3. Gene therapy can replace the defective gene with healthy gene
    1. Gene supplementation, several healthy genes are placed alongside the defective gene and the dominant healthy gene will be expressed
  4. Delivering healthy genes to sufferers:
    1. 1) Use of an adenovirus
      1. a) Virus rendered harmless by removal of some genes
        b) Virus grown in an epithelial cell culture together with plasmids and healthy gene
        c) Plasmids enter the adenovirus DNA
        d) Virus are extracted from the cell culture and inserted into the noses of patients
        e) The virus injects its DNA into the epithelial cells of the lungs
      2. Effects are short lived, can cause infections, patients can develop immunity to adenovirus, genes not always expressed
      3. Viruses can target specific cells and can adept their own methods of inserting into DNA
      4. With more treatments, there is a better immune response, so it is less likely to be taken up
        Antibodies may attack the virus
    2. 2) Use of a liposome
      1. Liposome aerosol may not be fine enough to pass through bronchioles
      2. CFTR gene
        Gene in vector
        Multiple copies are made of vector and insert
        Cloned copies of the vector placed into liposome
        Liposomes passed into lungs via an aerosol
        This makes the plasmid lipid soluble
      3. Very few of the genes are expressed
DNA probes
1. This is a:
  1. Short sequence of DNA
  2. Complementary nucleotide sequence of gene of interest
  3. Radioactive or fluorescence label added
2. Radioactive probe causes film to have a dark shadow where the radioactive label has been
3. Process of locating a specific allele of a gene:
  1. Sequence of nucleotides on mutated gene is determined by DNA sequencing
    1. Fragment of DNA with complementary bases to the mutant allele of the gene is produced
      1. DNA probe is formed by fluorescently labelling the DNA fragment
        PCR techniques are used to produce multiple copes of DNA probe
        Probe is added to single stranded DNA fragments from the person being screened
        If the donor has a mutated gene, some donor DNA fragments will be complementary to the probe, so the probe will bind
        These DNA fragments will now be labelled with the probe and can be distinguished
        If complementary fragments are present, the DNA probe will be taken up and the dye will fluoresce.
4. The more binding with the probe and the DNA (so hence more fluorescence) shows that more of that gene is present
  1. e.g.
    1. Where the probe is complementary to cystic fibrosis (allele = a)
    2. Aa
      1. aa
        AA
    3. Aa = carrier aa = disease AA = healthy
5. Screening for tumour suppressor gene is important because:
  1. Mutations of both alleles will result in inactive tumour suppressor
  2. Screening may identify hereozygous individuals for gene, so higher chance
6. This allows for personalised medicines:
  1. Medicinal treatments can be based on genotype
  2. Some patients genotype means drugs may be more/less effective
  3. Tailer drug selection and dosage to each individual
7. Genetic counselling is the process by which patients at risk of an inherited disorder are advised of the consequences and probability of having children
DNA fingerprinting
1. Some parts of non-coding DNA contain short sequences of repeated DNA
2. The technique:
  1. Extraction - DNA extracted from sample
    1. Digestion - restriction endonucleases cut the DNA into fragments
      1. Separation - Fragments separated with electrophoresis
        Separation - DNA fragments transferred from gel to nylon membrane
        Hybridisation - DNA probes added to label fragments
        Development - Membrane with radioactive labelled DNA fragments placed onto film
        Development - Reveals dark bands where DNA probes attached
3. Red blood cells cannot be used for DNA fingerprinting because they have no nucleus
4. DNA must be washed with dilute acid after the fragments are on the nylon membrane to make it single stranded
  1. This is because it must be single stranded to allow the probe to bind
Describing how genetic fingerprinting works in an exam:
1. 1) DNA is cut
  1. 2) Using restriction enzyme
    1. 3) Electrophoresis
      1. 4) Separates according to size
        5) DNA made single stranded
        6) Transfer to nylon membrane
        7) apply probe
        8) radioactivity detected on film
        9) Pattern is unique to every individual

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Pack 15 - Recombinant DNA technology

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	Created by Jacob Shepherd over 7 years ago